Pretreatment process for conversion of cellulose to fuel ethanol

ABSTRACT

An improved pretreatment of cellulosic feedstocks, to enable economical ethanol production by enzyme treatment. The improved pretreatment comprises choosing either a feedstock with a ratio of arabinoxylan to total nonstarch polysaccharides (AX/NSP) of greater than about 0.39, or a selectively bred feedstock on the basis of an increased ratio of AX/NSP over a starting feedstock material, and reacting at conditions that disrupt the fiber structure and hydrolyze a portion of the cellulose and hemicellulose. This pretreatment produces a superior substrate for enzymatic hydrolysis, by enabling the production of more glucose with less cellulase enzyme than any known procedures. This pretreatment is uniquely suited to ethanol production. Preferred feedstocks with an AX/NSP level greater than about 0.39 include varieties of oat hulls and corn cobs.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the production of fuel alcohol from cellulose.More specifically, this invention relates to the pretreatment ofcellulose feedstocks for ethanol production. The pretreatment reactionof feedstocks chosen with a ratio of arabinan plus xylan to non-starchpolysaccharides (AX/NSP) of greater than about 0.39 produces a superiorsubstrate for enzymatic hydrolysis than other feedstocks. Thesepretreated feedstocks are uniquely suited to ethanol production.Examples of feedstocks that could be chosen in such a pretreatmentprocess include some varieties of oat hulls and corn cobs, andfeedstocks selectively bred for high AX/NSP.

2. Brief Description of the Prior Art

The possibility of producing ethanol from cellulose has received muchattention due to the availability of large amounts of feedstock, thedesirability of avoiding burning or landfilling the materials, and thecleanliness of the ethanol fuel. The advantages of such a process forsociety are described, for example in a cover story of the ATLANTICMONTHLY, (April 1996).

The natural cellulosic feedstocks for such a process typically arereferred to as "biomass`. Many types of biomass, including wood,agricultural residues, herbaceous crops, and municipal solid wastes,have been considered as feedstocks for ethanol production. Thesematerials primarily consist of cellulose, hemicellulose, and lignin.This invention is concerned with converting the cellulose to ethanol.The familiar corn starch-to-ethanol process, in which the starch isconverted to ethanol using sulfurous acid and amylase enzymes, liesoutside the scope of this invention.

Cellulose is a polymer of the simple sugar glucose connected by beta 1,4linkages. Cellulose is very resistant to degradation or depolymerizationby acid, enzymes, or micro-organisms. Once the cellulose is converted toglucose, the resulting sugar is easily fermented to ethanol using yeast.The difficult challenge of the process is to convert the cellulose toglucose.

The oldest methods studied to convert cellulose to glucose are based onacid hydrolysis (review by Grethlein, Chemical Breakdown Of CellulosicMaterials, J.APPL.CHEM. BIOTECHNOL. 28:296-308 (1978)). This process caninvolve the use of concentrated or dilute acids. The concentrated acidprocess uses 72%, by weight, sulfuric acid or 42%, by weight,hydrochloric acid at room temperature to dissolve the cellulose,followed by dilution to 1% acid and heating to 100° C. to 120° C. for upto three hours to convert cellulose oligomers to glucose monomers. Thisprocess produces a high yield of glucose, but the recovery of the acid,the specialized materials of construction required, and the need tominimize water in the system are serious disadvantages of this process.Similar problems are encountered when concentrated organic solvents areused for cellulose conversion.

The dilute acid process uses 0.5% to 2%, by weight, sulfuric acid at180° C. to 240° C. for several minutes to several hours. BRINK (U.S.Pat. Nos. 5,221,537 and 5,536,325) describes a two-step process for theacid hydrolysis of lignocellulosic material to glucose. The first (mild)step depolymerizes the hemicellulose to xylose and other sugars. Thesecond step depolymerizes the cellulose to glucose. The low levels ofacid overcome the need for chemical recovery. However, the maximumglucose yield is only about 55% of the cellulose, and a high degree ofproduction of degradation products can inhibit the fermentation toethanol by yeast. These problems have prevented the dilute acidhydrolysis process from reaching commercialization.

To overcome the problems of the acid hydrolysis process, celluloseconversion processes have been developed using two steps: (1) apretreatment, and (2) a treatment comprising enzymatic hydrolysis. Thepurpose of pretreatment is not to hydrolyze the cellulose completely toglucose but, rather, to break down the integrity of the fiber structureand make the cellulose more accessible to attack by cellulase enzymes inthe treatment phase. After a typical pretreatment of this type, thesubstrate has a muddy texture. Pretreated materials also look somewhatsimilar to paper pulp, but with shorter fibers and more apparentphysical destruction of the feedstock.

The goal of most pretreatment methods is to deliver a sufficientcombination of mechanical and chemical action, so as to disrupt thefiber structure and improve the accessibility of the feedstock tocellulase enzymes. Mechanical action typically includes the use ofpressure, grinding, milling, agitation, shredding,compression/expansion, or other types of mechanical action. Chemicalaction typically includes the use of heat (often steam), acid, andsolvents. Several known pretreatment devices will be discussed below,and with specific reference to extruders, pressurized vessels, and batchreactors.

A typical treatment by enzymatic hydrolysis is carried out by mixing thesubstrate and water to achieve a slurry of 5% to 12%, by weight ofcellulose, and then adding cellulase enzymes. Typically, the hydrolysisis run for 24 to 150 hours at 50° C., pH 5. At the end of thehydrolysis, glucose, which is water soluble, is in the liquid whileunconverted cellulose, lignin, and other insoluble portions of thesubstrate remain in suspension. The glucose syrup is recovered byfiltering the hydrolysis slurry; some washing of the fiber solids iscarried out to increase the yield of glucose. The glucose syrup is thenfermented to ethanol by yeast, and the ethanol recovered by distillationor other means. The ethanol fermentation and recovery are bywell-established processes used in the alcohol industry.

The two-step process of pretreatment plus enzyme hydrolysis overcomesmany of the problems associated with a single harsh acid hydrolysis. Thespecific action of the enzymes decreases the amount of degradationproducts and increases the yield of glucose. In addition, the fact thatthe pretreatment for fiber destruction is milder than that for cellulosehydrolysis means that lower chemical loadings can be used, decreasingthe need for chemical recovery, and a lower amount of degradationproducts are made, increasing the yield and decreasing the inhibition offermentation to ethanol by yeast.

Unfortunately, to date the approach of a pretreatment and an enzymehydrolysis treatment has not been able to produce glucose at asufficiently low cost, so as to make a fermentation to ethanolcommercially attractive. Even with the most efficient currently knownpretreatment processes, the amount of cellulase enzyme required toconvert the cellulose to glucose is so high as to be cost-prohibitivefor ethanol production purposes.

Several approaches have been taken to attempt to decrease the amount ofcellulase enzyme required.

The approach of simply adding less cellulase to the system decreases theamount of glucose produced to an unacceptable extent.

The approach of decreasing the amount of enzyme required by increasingthe length of time that the enzyme acts on the feedstock leads touneconomical process productivity, stemming from the high cost ofhydrolysis tanks.

The approach of reducing the amount of cellulase enzyme required bycarrying out cellulose hydrolysis simultaneously with fermentation ofthe glucose by yeast is also inefficient. The so-called simultaneoussaccharification and fermentation (SSF) process is not yet commerciallyviable because the optimum operating temperature for yeast, 28° C., istoo far below the optimum 50° C. conditions required by cellulase.Operating a SSF system at a compromise temperature of 37° C. is alsoinefficient, and invites microbial contamination.

The desire for a cost-effective ethanol production process has motivateda large amount of research into developing effective pretreatmentsystems. Such a pretreatment system would achieve all of the advantagesof current pretreatments, including low production of degradationproducts and low requirements for chemical recovery, but with asufficiently low requirement for cellulase enzymes so as to make theprocess economical.

The performance of a pretreatment system is characterized strictly bythe amount of enzyme required to hydrolyze an amount of cellulose toglucose. Pretreatment A performs better than pretreatment B, if Arequires less enzyme to produce a given yield of glucose than B.

The early work in pretreatment focused on the construction of a workingdevice and determination of the conditions for the best performance.

One of the leading approaches to pretreatment is by steam explosion,using the process conditions described by FOODY (U.S. Pat. No.4,461,648), which is incorporated herein by reference. In the FOODYprocess, biomass is loaded into a vessel known as a steam gun. Up to 1%acid is optionally added to the biomass in the steam gun or in apresoak. The steam gun is then filled very quickly with steam and heldat high pressure for a set length of time, known as the cooking time.Once the cooking time elapses, the vessel is depressurized rapidly toexpel the pretreated biomass, hence the terminology "steam explosion"and "steam gun".

In the FOODY process, the performance of the pretreatment depends on thecooking time, the cooking temperature, the concentration of acid used,and the particle size of the feedstock. The recommended pretreatmentconditions in the FOODY process are similar for all the cellulosicfeedstocks tested (hardwood, wheat straw, and bagasse) provided they aredivided into fine particles. Furthermore, the cooking temperature isdetermined by the pressure of the saturated steam fed into the steamgun. Therefore, the practical operating variables that effect theperformance of the pretreatment are the steam pressure, cooking time,and acid concentration. The FOODY process describes combinations ofthese variables for optimum performance; as one might expect, increasingthe time decreases the temperature used, and vice versa. The range ofsteam pressure taught by FOODY is 250 psig to 1000 psig, whichcorresponds to temperatures of 208° C. to 285° C.

Another published study of steam explosion pretreatment parameters isFoody, et al, Final Report, Optimization of Steam ExplosionPretreatment, U.S. DEPARTMENT OF ENERGY REPORT ET230501 (April 1980).This study reported the effects of the pretreatment variables oftemperature (steam pressure), particle size, moisture content,pre-conditioning, die configuration, and lignin content. The optimizedsteam explosion conditions were reported for three types of straws, fivespecies of hardwood, and four crop residues.

The optimum pretreatment conditions as published by FOODY weresubsequently confirmed by others using other feedstocks and differentequipment. For example, GRETHLEIN (U.S. Pat. No. 4,237,226), describespretreatment of oak, newsprint, poplar, and corn stover by a continuousplug-flow reactor, a device that is similar to an extruder. Rotatingscrews convey a feedstock slurry through a small orifice, wheremechanical and chemical action break down the fibers.

GRETHLEIN specifies required orifice sizes, system pressures,temperatures (180° C. to 300 C.), residence times (up to 5 minutes),acid concentrations (up to 1% sulfuric acid) and particle sizes(preferred 60 mesh). GRETHLEIN obtained similar results for all of thespecified substrates he identified (See Column 3, line 30). Even thoughthe GRETHLEIN device is quite different from the steam gun of FOODY, thetime, temperature, and acid concentration for optimum performance aresimilar.

More recent work has focused on understanding the means by whichpretreatment improves the enzymatic hydrolysis of a given substrate.BRINK (U.S. Pat. No. 5,628,830) describes the pretreatment oflignocellulosic material by using a steam process to break down thehemicellulose and following with hydrolysis of the cellulose usingcellulase enzymes.

The first explanation offered to characterize the advantage of apretreatment was that a pretreatment should be evaluated on the amountof lignin removed, with better performance associated with higherdegrees of delignification. See Fan, Gharpuray, and Lee, Evaluation OfPretreatments For Enzymatic Conversion Of Agricultural Residues,PROCEEDINGS OF THE THIRD SYMPOSIUM ON BIOTECHNOLOGY IN ENERGY PRODUCTIONAND CONSERVATION, (Gatlinburg, Tenn., May 12-15, 1981). The notion thatdelignification alone characterizes pretreatment was also reported byCunningham, et al, PROCEEDINGS OF THE SEVENTH SYMPOSIUM ON BIOTECHNOLOGYFOR FUELS AND CHEMICALS, (Gatlinburg, Tenn., May 14-17, 1985).

Grethlein and Converse, Common Aspects of Acid Prehydrolysis and SteamExplosion for Pretreating Wood, BIORESOURCE TECHNOLOGY 36(2):77-82(1991), put forth the proposition that the degree of delignification isimportant only for previously dried substrates and, therefore, not arelevant consideration to most pretreatment processes that use undriedfeedstocks.

Knappert, et al, A Partial Acid Hydrolysis of Cellulosic Materials as aPretreatment for Enzymatic Hydrolysis, BIOTECHNOLOGY AND BIOENGINEERING23:1449-1463 (1980) reported that the increased susceptibility to enzymehydrolysis after pretreatment is caused by the creation of micropores bythe removal of the hemicellulose, a change in crystallinity of thesubstrate, and a gross reduction in the degree of polymerization of thecellulose molecule.

Grohmann, et al, Optimization of Dilute Acid Pretreatment of Biomass,SEVENTH SYMPOSIUM ON BIOTECHNOLOGY FOR FUELS AND CHEMICALS (Gatlinburg,Tenn., May 14-17, 1985) specifically supported one of the hypotheses ofKnappert, et al by showing that removal of hemicellulose in pretreatmentresults in improved enzymatic hydrolysis of the feedstock. (See p.59-80). Grohmann, et al worked with wheat straw and aspen wood attemperatures of 95° C. to 160° C. and cooking times of up to 21 hours.For both feedstocks, about 80% of the cellulose was digested bycellulase enzymes after optimum pretreatments, in which 80% to 90% ofthe xylan was removed from the initial material.

Grohmann and Converse also reported that the crystallinity index of thecellulose was not changed significantly by pretreatment. They furtherreported that pretreatments can create a wide range of degrees ofpolymerization while resulting in similar susceptibility to enzymatichydrolysis.

Another alternative explanation offered for the improvements inenzymatic hydrolysis due to pretreatment is the increase in surface areaof the substrate. Grethlein and Converse refined this explanation byshowing that the surface area that is relevant is that which isaccessible to the cellulase enzyme, which has a size of about 51angstroms. The total surface area, which is measured by theaccessibility of small molecules such as nitrogen, does not correlatewith the rate of enzymatic hydrolysis of the substrate, for the reasonthat small pores that do not allow the enzyme to penetrate do notinfluence the rate of hydrolysis.

In spite of a good understanding of devices and optimum conditions forpretreatment, and a large quantity of research into the mechanism of apretreatment process, there still does not exist an adequatepretreatment for a commercially feasible process to convert cellulosicmaterials to ethanol. Such a pretreatment process would be of enormousbenefit in bringing the cellulose-to-ethanol process to commercialviability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A graph of cellulose conversion for certain feedstocks afterpretreatment reaction at 121 C., as a function of AX/NSP of the initialmaterial, according to EXAMPLE 3.

FIG. 2 A graph of cellulose conversion for certain feedstocks afterpretreatment reaction at 230 C., as a function of AX/NSP of the initialmaterial, according to EXAMPLE 4.

SUMMARY OF THE INVENTION

The inventors have discovered that a critical property of a feedstockdetermines its relative cellulase enzyme requirement to convert thecellulose to glucose after the pretreatment reaction. That property isthe ratio of arabinan plus xylan to total nonstarch polysaccharides,which we will refer to hereinafter as "AX/NSP." The inventors havediscovered that the higher the AX/NSP, the less cellulase enzyme isrequired after the pretreatment reaction, and hence the more economicalthe production of ethanol. Feedstocks with AX/NSP over about 0.39 areparticularly well suited for a cellulose-to-ethanol process. Examples ofsuch feedstocks are certain varieties of oat hulls and corn cobs.

Based on this discovery, the inventors have developed improvedpretreatment processes prior to enzyme treatment that converts alignocellulosic feedstock to ethanol. One such process consistsessentially of the steps:

1. Choosing a lignocellulosic feedstock with a ratio of arabinan plusxylan to total nonstarch polysaccharides (abbreviated AX/NSP) of greaterthan 0.39.

2. Reacting the chosen feedstock at conditions which disrupt the fiberstructure and effect an hydrolysis upon portions of both cellulose andhemicellulose, so as to improve digestibility of the pretreatedfeedstock by a subsequent cellulase enzyme treatment.

A second such process consists essentially of the steps:

1. Choosing a lignocellulosic feedstock selectively bred to have arelatively increased ratio of arabinan plus xylan to total nonstarchpolysaccharides (AX/NSP).

2. Reacting the chosen feedstock at conditions which disrupt the fiberstructure and effect an hydrolysis upon portions of both cellulose andhemicellulose, so as to improve digestibility of the pretreatedfeedstock by a subsequent cellulase enzyme treatment.

Once a feedstock is chosen based on high AX/NSP, the pretreatmentreactions can be carried out in a manner consistent with previousreports. This might include single stage or two stage reactions in steamguns, extruders, or other devices used previously.

By choosing the feedstock based on AX/NSP, the resulting cellulaseenzyme requirement after the pretreatment reaction is significantlylower than otherwise required. This results in significant savings inthe cost of producing ethanol from lignocellulosic materials.

There have been no previous reports of the superior performance afterpretreatment of feedstocks specifically chosen because of any particularlevel of AX/NSP, let alone an AX/NSP level that is greater than 0.39, byweight.

The present invention is very surprising in view of the U.S. D.O.E.study by FOODY, et al, supra, which observed no correlation betweenxylan content of the feedstocks and glucose yield after steam explosionand hydrolysis by cellulase.

FOODY, et al was a study of thirteen feedstocks. The resultingconversion of cellulose to glucose varied widely among the pretreatedfeedstocks, between 46% to 50% for oak and sunflower stalks to 86% to87% for barley straw and maple. The two best feedstocks of FOODY et al,barley straw and maple wood, had xylan contents of 31% and 19%,respectively, which were among the highest and lowest values reported.Oak and aspen both contained 21% xylan, yet they achieved widelydiffering glucose yields after hydrolysis by cellulase, 46% and 72%,respectively.

The present invention also is very surprising in view of the patent toGRETHLEIN, supra. GRETHLEIN described a device for the pretreatment offeedstocks using dilute sulfuric acid. All four of the GRETHLEINfeedstocks (oak, newsprint, poplar, and corn stover) performed similarly(Column 3, lines 25 to 32). This reported result is exactly contrary tothe teachings of the present invention, who have found and identified anovel feedstock property, AX/NSP, that can reliably be used to predictthe performance of the feedstocks after treatment.

The present invention also is very surprising in view of the publicationby Knappert, et al., supra, which reviewed four feedstocks: Solka floc,newsprint, oak, and corn stover. Knappert, et al obtained optimum yieldsof glucose from cellulose after pretreatment reactions. One hundredpercent yield was obtained from newsprint, corn stover, and oak, and 81%yield was obtained from Solka floc (Tables I and II, page 1453-1457). Asthe only feedstocks with cellulose and xylan content reported werenewsprint and Solka floc, this study simply does not address therelationship between AX/NSP of the feedstock and the digestibility ofthe material by cellulase enzymes after pretreatment reaction.

The present invention actually suggests that the teachings of Knappert,et al are incorrect. At the very least, the teachings of Knappert, et alare at odds with the teachings of the present invention. Knappert et altaught that a low hemicellulose content of a material presages littleimprovement in cellulose digestibility during pretreatment. The presentinvention, at EXAMPLE 5, shows a large improvement in the digestibilityof oat hulls with pretreatment reaction after the hemicellulose has beenremoved by a mild reaction.

SUMMARY OF TERMINOLOGY

The invention and preferred embodiments described hereafter are to beconstrued using certain terms as hereafter defined, for purposes of thepresent invention.

Lignocellulosic feedstock means any raw material that one might considerfor a cellulose-to-ethanol process. Such a material has at least about25% cellulose, and the cellulose is substantially converted to glucoseand then ethanol in the process. Typical lignocellulosic feedstocksmaterials are wood, grains, and agricultural waste. For the presentpurposes there are no specifications on the lignin, starch, protein, orash content. Examples of lignocellulosic feedstocks that have beenconsidered for an ethanol process are wood, grasses, straws, and cropwaste. Often, a lignocellulosic feedstock originates from one species offiber. However, for present purposes the lignocellulosic feedstock canbe a mixture that originates from a number of different species.

Conversion to fuel ethanol denotes the conversion of at least about 40%of the cellulose to glucose, and then fermentation of the glucose toethanol. For the present purposes there are no specifications on theconversion products made from the lignin or the hemicellulose. In apreferred embodiment, at least 60% of the cellulose is converted toglucose and fermented to ethanol.

Xylan and xylan content are the terms used to express the quantity ofanhydroxylose present in the feedstock. Much of the anhydroxylose ispresent as a linear beta 1,4-linked polysaccharide of xylose, but thedesignation xylan is not limited to anhydroxylose of this structure.

Arabinan and arabinan content are the terms used to express the quantityof anhydroarabinose present in the feedstock. Much of theanhydroarabinose is present as a branched alpha 1,3-linkedpolysaccharide of arabinose, but the designation arabinan is not limitedto anhydroarabinose of this structure.

Arabinan plus xylan refers to the sum of the arabinan content and thexylan content of the feedstock. This is distinguished from the termarabinoxylan, which refers to an alpha 1,3-linked polymer of arabinoseand xylose. Arabinoxylan is a specific example of arabinan and xylan,but does not comprise all possible forms of arabinan and xylan.

Hemicellulose is a general term that includes all naturalpolysaccharides except cellulose and starch. The term includes polymersof xylose, arabinose, galactose, mannose, etc. and mixtures thereof. Inthe present work, the primary constituents of the hemicellulose arearabinose and xylose.

AX/NSP is the ratio of arabinan plus xylan to non-starch polysaccharidesand can be measured for any feedstock based on the analytical proceduresdescribed herein. AX/NSP is calculated from EQUATION (1):

    AX/NSP=(xylan+arabinan)/(xylan+arabinan+cellulose)         (1)

where the xylan, arabinan, and cellulose contents of the feedstocks aremeasured according to the procedures in EXAMPLE 1 and AX/NSP iscalculated as shown in EXAMPLE 1.

AX/NSP is taught herein to characterize the performance of thepretreatment. The higher the AX/NSP, the less cellulase enzyme isrequired to hydrolyze the cellulose to glucose after a givenpretreatment. The pretreatment performance is particularly good forfeedstocks with AX/NSP of greater than about 0.39. This point isillustrated in EXAMPLES 3 and 4.

The AX/NSP content should be measured for each batch of a feedstockused, as it will no doubt vary seasonally and with the age, geographicallocation, and cultivar of the feedstock. Therefore, there are noabsolute values of AX/NSP that are always valid for a given species.However, samples of oat hulls and corn cobs exhibited the highest AX/NSPin the data collected, as well as the highest performance inpretreatment. Oat hulls and corn cobs from the lots sampled wouldtherefore be preferred feedstocks for an ethanol process.

The theoretical upper limit of AX/NSP is 0.75. This would be present ina material that was 25% cellulose and 75% arabinan plus xylan. Theinventors know of no materials with this composition. The highest AX/NSPobserved by the inventors is 0.422.

The hemicellulose, cellulose, arabinan, and xylan content of variousmaterials have been widely published. However, the analytical methodsused can greatly influence the apparent composition, and thesepublications are often based on widely varying methods. Therefore, thesepublications can be relied on only to give a general idea as to theapproximate composition of these materials. For the purposes ofpracticing the invention, the same analytical methods must be applied toeach candidate feedstock, and those of Example 1 are preferred for theabsolute values being claimed.

In practicing the invention, feedstocks with high AX/NSP can beidentified by two generic methods: (1) by screening of natural fibersand grains, and (2) by screening of varieties selectively bred forhigher AX/NSP levels.

Reaction or Pretreatment reaction refers to a chemical process used tomodify a lignocellulosic feedstock to make it more amenable tohydrolysis by cellulase enzymes. In the absence of pretreatment, theamount of cellulase enzyme required to produce glucose is impractical.

Improve digestibility by cellulase enzymes by disrupting the fiberstructure and effecting the hydrolysis of a portion of the hemicelluloseand the cellulose. This terminology refers to the physical and chemicalchanges to the feedstock caused by the pretreatment reaction. At aminimum, pretreatment increases the amount of glucose hydrolyzed fromthe feedstock by cellulase, disrupts the fibers, and hydrolyzes somefraction of the cellulose and hemicellulose.

The pretreatment process of the invention preferably is part of anintegrated process to convert a lignocellulosic feedstock to ethanol.Such a process includes, after pretreatment, enzymatic hydrolysis ofcellulose to glucose, fermentation of the glucose to ethanol, andrecovery of the ethanol.

Cellulose hydrolysis refers to the use of cellulase enzymes to convertthe pretreated cellulose to glucose. In the present invention, aminority of the cellulose is hydrolyzed during the pretreatment, and themajority survives pretreatment and is subjected to hydrolysis bycellulase enzymes. The manner in which the enzymatic hydrolysis iscarried out is not constrained by the invention, but preferredconditions are as follows. The hydrolysis is carried out in a slurrywith water that is initially 5% to 12% cellulose and is maintained at pH4.5 to 5.0 and 50° C. The cellulase enzymes used might be any of thecommercial cellulases available, which are manufactured by IOGENCORPORATION, Novo NORDISK, GENENCOR INTERNATIONAL, PRIMALCO, and othercompanies. The cellulase enzymes might be supplemented withbeta-glucosidase to complete the conversion of cellobiose to glucose. Acommercial beta-glucosidase enzyme is NOVOZYM 188, sold by Novo NORDISK.

The skilled practitioner will realize that the amount of cellulaseenzyme used in the hydrolysis is determined by the cost of the enzymeand the desired hydrolysis time, glucose yield, and glucoseconcentration, all of which are influenced by the process economics andwill vary as each of the relevant technologies is evaluated. The typicalenzyme dosage range is 1 to 50 Filter Paper Units (FPU) cellulase pergram cellulose for 12 to 128 hours. In a preferred embodiment thecellulase enzyme dosage is 1 to 10 FPU per gram cellulose. EXAMPLES 2and 3 describe cellulose hydrolysis in more detail.

In a preferred embodiment, cellulose hydrolysis and ethanol fermentationare carried out simultaneously, using those techniques generallyemployed in an SSF process, as discussed previously herein.

Ethanol fermentation and recovery are carried out by conventionalprocesses that are well known, such as yeast fermentation anddistillation. The invention is not constrained by the manner in whichthese operations are carried out.

DESCRIPTION OF PREFERRED EMBODIMENTS

In practicing the invention, any type of feedstock, including but notlimited to naturally occurring and selectively bred feedstock, can beemployed. As emphasized above, the novelty of the present inventionrelates to the use of a high AX/NSP ratio, heretofore unrecognized as acritical standard for choosing optimum feedstocks for glucose andethanol production; the origin of the feedstock is of secondaryimportance.

In one embodiment, the feedstock is naturally occurring. In this case,the AX/NSP of the feedstock is measured by the method of Example 1.Feedstocks with AX/NSP of greater than about 0.39 are preferred for acellulose-to-ethanol process.

The AX/NSP content should be measured for each batch of a feedstockused, as it will no doubt vary seasonally and with age, geographiclocation, and cultivar of the feedstock. As experience with a givenfeedstock accumulates, the frequency of testing AX/NSP will lessen.

In another preferred embodiment, the feedstock has already beenselectively bred. In this case, the AX/NSP of the bred feedstock ismeasured by the method of Example 1 and compared with that of thenatural feedstock. If the AX/NSP has been increased by breeding, thefeedstock is more suitable for cellulose conversion than the natural orstarting feedstock material.

Such breeding can, in principle, be carried out by any of the commonmethods used to select for desired traits in plant breeding. Thesemethods are summarized by H. B. Tukey, "Horticulture is a Great GreenCarpet that Covers the Earth" in American Journal of Botany44(3):279-289 (1957) and Ann M. Thayer, "Betting the Transgenic Farm" inChemical and Engineering News, Apr. 28, 1997, p. 15-19. The methodsinclude:

1. Scientific Breeding. Screen varieties of a species for a high levelof AX/NSP and repeatedly grow those varieties which exhibit the trait.

2. Chimaeras. Graft two or more species and screen the resulting speciesfor the level of AX/NSP.

3. Pollination breeding. Combine two or more species by crosspollination and screen for AX/NSP level.

4. Chemical thinning. Expose plants to chemical toxins such that onlythe fittest survive. Requires a toxin that is resisted by arabinan orxylan.

5. Induction. Expose species to conditions that induce higher levels ofAX/NSP.

6. Environmental distress. Expose species to conditions that inducedeath unless protested by high levels of AX/NSP.

7. Nutrition and fertilizers. Develop nutritional regimen to increaseAX/NSP.

8. Genetic engineering. Genetically modify a species so as to increaseits level of AX/NSP.

In one preferred embodiment, the selectively bred lignocellulosicfeedstock has an AX/NSP level that is greater than about 0.39, and sucha selectively bred feedstock then is reacted to increase itsdigestibility by cellulase enzymes and converted to ethanol byhydrolyzing the cellulose to glucose with cellulase enzymes, fermentingthe glucose, and recovering the ethanol.

In another preferred embodiment, the selectively bred lignocellulosicfeedstock has an increased AX/NSP level over a starting feedstockmaterial, but still below 0.39. Such a selectively bred feedstock isthen reacted to increase its digestibility to cellulase enzymes andconverted to ethanol. The reason that increasing the AX/NSP content of afeedstock is beneficial, even if the level remains below 0.39, is thatin certain geographical areas the climate supports the growth of only anarrow range of feedstocks. For example, corn does not grow in climateswhere the annual number of degree days above 40 F. is less than 240. Inthese cooler areas, the choice of feedstocks is limited, and there mightnot be any feedstocks available with AX/NSP close to 0.39. In theseclimates, improving such a feedstock by selectively breeding to increaseits AX/NSP over a starting feedstock material would improve theefficiency of a cellulose-to-ethanol plant significantly, even if theAX/NSP still remained below 0.39. In these situations, the presentinvention would provide a novel standard against which such selectivelybred feedstocks could be measured and compared.

The desired extent of pretreatment might be achieved by any meansavailable, including but not limited to those discussed in the preferredembodiments or examples contained herein. Any combination of mechanicaland chemical treatments that results in the chemical changes noted lieswithin the scope of practicing the invention. This includes anyreactors, chemicals added, temperature, time, particle size, moisture,and other parameters that result in the changes to the feedstock.

In a first preferred embodiment, the pretreatment reaction is carriedout at the broad conditions described by GRETHLEIN for acidpretreatments. This is done by subjecting the chosen feedstock to atemperature of about 180° C. to about 270° C., for a period of 5 secondsto 60 minutes. It is understood by those skilled in the art that thefeedstock temperature is that of the feedstock itself, which mightdiffer from the temperature measured outside the reaction chamber. It isalso understood by those skilled in the art that a temperature rangespecified over a time period is the average temperature for that period,taking into account the effect of temperature on the rate of reaction.For example, the reaction chamber might require a short period to heatfrom ambient conditions up to 180° C. Based on knowledge of reactionkinetics (for example, within limited temperature ranges for a givensubstance, the rate approximately doubles over a 10° C. increase intemperature), the effect of the temperature increase on the overallreaction can be calculated and thereby the average temperaturedetermined.

The pretreatment reaction is typically run with 0.1% to 2% sulfuric acidpresent in the hydrolysis slurry. However, those skilled in the art areaware that alkali or acid present in some feedstocks can alter the acidrequirement to be outside of the typical range. The degree of aciditypresent is better expressed by the target pH range, which is 0.5 to 2.5regardless of the acid or concentration used. EXAMPLE 8 illustratespretreatment reactions at this range of conditions.

A second preferred embodiment uses the narrower set of conditionsidentified by FOODY as optimal for steam explosion pretreatment. This isillustrated in EXAMPLE 4 with pretreatment consisting of a cooking stepat a temperature between 220° C. to 270° C. at pH 0.5 to 2.5 for 5seconds to 120 seconds. Devices used to carry out this pretreatmentpreferably include sealed batch reactors and continuous extruders. Largescale examples of these pretreatment conditions are described inEXAMPLES 6 and 7.

A third preferred embodiment uses a two-stage pretreatment, whereby thefirst stage improves the cellulose hydrolysis somewhat whilesolubilizing primarily the hemicellulose but little cellulose. Thesecond stage then completes a full pretreatment. In this embodiment, thefirst stage reaction is run at a temperature of less than 180° C. whilethe second stage reaction is run at a temperature of greater than 180°C. An advantage of a two-stage pretreatment, as shown hereafter inEXAMPLE 5, is that a separate recovery of the hemicellulose fordownstream processing is facilitated.

In the third preferred embodiment, the first stage of reaction iscarried out at a temperature of about 60° C. to about 140° C. for 0.25to 24 hours at pH 0.5 to 2.5. More preferably, the first stage ofpretreatment is carried out at a temperature of 100° C. to 130° C. for0.5 to 3 hours at pH 0.5 to 2.5.

In the fourth preferred embodiment, the second stage of reaction iscarried out at a temperature of 180° C. to 270° C., at pH 0.5 to 2.5 fora period of 5 seconds to 120 seconds. The feedstock also can be dry(free from added moisture) or in a slurry with water.

In a preferred embodiment, the selectively bred feedstock is a woodyfiber. W ood is the most prevalent lignocellulosic material in coolerclimates.

Another aspect to successful practice of the present invention is tointegrate the pretreatment process within a process that hydrolyzes thepretreated feedstock with cellulase enzymes to produce glucose. In apreferred embodiment, at least 40% of the cellulose in the pretreatedfeedstock is hydrolyzed by cellulase enzymes to produce glucose. Theglucose produced can be purified, crystallized, and packaged as solidsugar. Alternatively, it can be left dissolved in a liquid slurry forfurther processing or use.

EXAMPLE 1 Measurement of AX/NSP in Feedstocks

The ratio of arabinan plus xylan to total non-starch polysaccharides ofa given feedstock was determined based on a composition al analysis ofthe feeds tocks. This analysis was performed, as follows.

Feedstocks examined were barley straw, wheat straw, wheat chaff, oathulls, switch grass, corn stover, maple wood, pine wood, and threevarieties of corn cobs. All were obtained locally in Ottawa, Ontarioexcept the oat hulls, which were from Quaker Oats in Peterborough,Ontario. The feedstocks were coarsely ground in a Waring blender andthen milled through a #20 gauge screen using a Wiley mill. Thefeedstocks were stored at ambient temperature in sealed bags until thetime of use. The moisture content of small samples was 5% to 10% and wasdetermined by drying at 100° C.

Approximately 0.3 grams of sample was weighed into test tubes, ea chcontaining 5 ml of 70% sulfuric acid. The tubes were vortex mixed,capped, and placed in a 50° C. water bath for one hour, with vigorousvortex mixing every 10 minutes. After the one hour incubation, the tubecontents were transferred into preweighed 250 ml flasks containing 195ml deionized water, which reduced the ac id content to 1.75%. Thecontents were mixed, and then 10 gram aliquots were transferred intotest tubes. The tubes were vortex mixed and then transferred to a steamautoclave, where they were maintained for 1 hour at 121° C. Afterautoclaving, the solution contents were neutralized using a small amountof barium carbonate, and then vacuum-filtered over glass microfiberfilter paper.

The concentrations of glucose, xylose, and arabinose present in thefiltrates were measured by using a Dionex Pulse-Amperometric HPLC. Thesemeasurements were then related to the weight of the initial sample offeedstock present and expressed as glucan, xylan, and arabinan contents,respectively, of the feedstock, with small adjustments to take intoaccount (1) the water of hydration to make the monomers from polymersand (2) the amount of material destroyed by the concentrated acid, whichwas measured by taking pure cellulose, xylose, and arabinose controlsthrough the procedure. The determination was performed in triplicate andthe average value is reported.

The cellulose content was determined by subtracting the starch contentfrom the total glucan. The starch content was determined by adding 1gram of Wiley-milled feedstock to a 250 ml flask containing 20 ml ofdeionized water, 0.2 ml of 91.7 g/L CaCl₂.2H₂ O stock solution, and 50microliters of a 1:100 solution of Sigma Alpha Amylase #A3403 indeionized water. Each flask was adjusted to pH 6.4 to 6.6 using dilutesodium hydroxide, then incubated in a boiling water bath for one hour.The flasks were incubated for 30 minutes in a steam autoclave at 121° C.after the addition of a second 50 ml dose of amylase. Finally, the flaskwas incubated for another 60 minutes in the boiling water bath with athird 50 ml dose of amylase. The flasks were then cooled to ambienttemperature and adjusted to pH 4.2 to 4.4 using dilute hydrochloricacid. A 0.5 ml aliquot of Novo Spritamylase stock solution was added;the stock solution consisted of 3 grams of enzyme in 100 ml deionizedwater. The flasks were shaken at 50° C. for 20 hours with 150 RPMagitation. The flasks were then cooled and the contents were filteredover glass microfiber filter paper. The glucose concentration was thenmeasured on a Yellow Springs Instrument (YSI) glucose analyzer and usedto determine the starch concentration of the feedstock, taking intoaccount the water necessary to hydrolize the starch.

The protein and ash content of the feedstocks were determined bystandard Kjeldahl nitrogen and ash oven methods.

The lignin content of the samples was determined by measuring the amountof insoluble solids remaining after the sulfuric acid treatment of thefeedstocks, then subtracting the amount of ash present.

The results of these measurements are shown in TABLE 1. The materialrecovered was between 842 and 1019 mg per gram of original solids(mg/g). This corresponds to 84.2%, by weight, to 101.9% of the startingmaterial, which is typical mass balance closure in these systems.

                                      TABLE 1    __________________________________________________________________________    COMPOSITION OF THE FEEDSTOCKS    Measured composition (mg/g)    Feedstock         Glucan             Starch                 Xylan                     Arabinan                          Lignin                               Ash                                  Protein                                      Total    __________________________________________________________________________    Barley         426 19.6                 161 28   168  82 64  929    Straw    Wheat         464 8.6 165 25   204  83 64  1005    Straw    Wheat         405 14.4                 200 36   160  121                                  33  955    chaff    Switch         403 3.4 184 38   183  48 54  910    grass    Corn 411 3.2 128 35   127  60 81  842    stover    Maple         504 4.0 150 5    276   6  6  947    wood    Pine 649 1.0 33  14   320   0  2  1018    wood    Corn cobs         436 34  253 38   .sup. ND.sup.(2)                               ND ND  ND    (red)    Corn cobs         439 28  250 38   ND   ND ND  ND    (white)    Corn cobs         438 8.5 240 36   ND   ND ND  ND    (Indian)    Oat Hulls         481 89  247 39   170  44 38  1019    __________________________________________________________________________     .sup.(1) Total = Glucan + Xylan + Arabinan + Lignin + Ash + Protein     .sup.(2) ND = Not determined

The AX/NSP content of the feedstocks is shown in TABLE 2. Of the 11feedstocks analyzed, four have AX/NSP of greater than about 0.39. Theseinclude the samples of oat hulls and corn. The other seven feedstockshave AX/NSP content below about 0.39.

                  TABLE 2    ______________________________________    AX/NSP COMPOSITION OF THE FEEDSTOCKS            Cellulose            NSP    Feed-stock            (mg/g).sup.(1)                      AX (mg/g).sup.(2)                                 (mg/g).sup.(3)                                         AX/NSP    ______________________________________    Barley  407       189        596     0.317    Straw    Wheat   455       190        645     0.295    Straw    Wheat   391       236        627     0.376    chaff    Switch  399       222        621     0.357    grass    Corn    408       163        571     0.285    stover    Maple   500       155        655     0.237    wood    Pine    648       47         695     0.068    wood    Corn cobs            402       291        693     0.420    (red)    Corn cobs            411       288        699     0.412    (white)    Corn cobs            429       276        705     0.391    (Indian)    Oat Hulls            392       286        678     0.422    ______________________________________     .sup.(1) Cellulose = Glucan  Starch     .sup.(2) AX = Xylan + Arabinan     .sup.(3) NSP = Xylan + Arabinan + Cellulose

EXAMPLE 2 Measurement of Cellulase Activity of an Enzyme

The cellulase activity of an enzyme is measured using the procedures ofGhose, PURE AND APPL. CIIEM., 59:257-268 (1987), as follows. A 50 mgpiece of Whatman #1 filter paper is placed in each test tube with 1 mlof 50 mM sodium citrate buffer, pH 4.8. The filter paper is rolled upand the test tube is vortex mixed to immerse the filter paper in theliquid. A dilution series of the enzyme is prepared with concentrationsranging between 1:200 and 1:1600 of the initial strength in 50 mM sodiumcitrate buffer, pH 4.8. The dilute enzyme stocks and the substrates areseparately preheated to 50° C., then a 0.5 ml aliquot of each diluteenzyme stock is placed in a test tube with substrate. The test tubes areincubated for 60 minutes at 50° C. The reaction is terminated by adding3 ml of dinitrosalicylic acid (DNS) reagent to each tube and thenboiling for 10 minutes. Rochelle salts and deionized water were added toeach tube to develop the color characteristic of the reaction betweenreducing sugars and DNS reagent. The amount of sugar produced by eachsample of enzyme is measured, taking into account the small backgroundfrom the enzyme and the filter paper, by comparing the amount of sugarin each tube with that of known sugar standards brought through thereaction.

A unit of filter paper activity is defined as the number of micromolesof sugar produced per minute. The activity is calculated using theamount of enzyme required to produce 2 mg of sugar. A sample of IogenCellulase was found to have 140 filter paper units per ml, as shown inTABLE 3.

                  TABLE 3    ______________________________________    FILTER PAPER ACTIVITY OF IOGEN CELLULASE    Amount of enzyme (ml)    to make 2 mg sugar                    Enzyme activity (FPU/ml)    ______________________________________    0.00264         140.0    ______________________________________

EXAMPLE 3 Mild Pretreatment Reaction with the Feedstocks

This example illustrates the comparative performance of the feedstocksafter a mild pretreatment reaction that primarily dissolves thehemicellulose. This pretreatment reaction by itself is not optimal,although it could be the first stage of a two-stage pretreatmentreaction. This mild reaction illustrates the use of AX/NSP tocharacterize the suitability of a feedstock for ethanol production.Optimized pretreatment reactions are described in later examples.

Samples of 4 grams of Wiley-milled feedstocks from EXAMPLE 1 were placedin 96 grams of 1% sulfuric acid (pH 0.6 to 0.9) in a 250 ml flask. Thecontents of the flasks were gently mixed, and then the flasks wereplaced in a steam autoclave at 121° C. for 1 hour. The flasks were thencooled and vacuum-filtered over glass microfiber filter paper. Theglucose, xylose, and arabinose concentrations of selected filtrates weredetermined by neutralizing with barium carbonate and analyzing thesamples using a Dionex Pulsed-Amperometric HPLC. The filter cakes werewashed with tap water and air dried. The cellulose, xylan, and arabinanconcentrations in the solids were determined by dissolution of aliquotsin 70% sulfuric acid, as described in EXAMPLE 1.

The effect of the reaction on the cellulose and hemicellulose levels inthe selected feedstocks is shown in TABLE 4. In all cases, small amounts(less than 8%) of the cellulose is hydrolyzed, while more than 70% ofthe hemicellulose is hydrolyzed.

                  TABLE 4    ______________________________________    EFFECT OF 121° C. PRETREATMENT REACTION ON    DIFFERENT FEFDSTOCKS    Dissolution (%)                            Hemi-    Feedstock      Cellulose                            cellulose    ______________________________________    Barley         3.2      85    straw    Wheat          3.6      72    straw    Wheat          <2       75    chaff    Switch         5.7      80    grass    Corn           4.3      82    stover    Maple          <2       80    wood    Oat hulls      7.9      85    ______________________________________

All 11 pretreated feedstocks were subjected to cellulase enzymehydrolysis as follows. A sample of the pretreated solids correspondingto 0.2 grams of cellulose was added to a 250 ml flask with 19.8 grams of0.05 M sodium citrate buffer, pH 5.0. Iogen Cellulase (standardized to140 FPU/ml) and Novozym 188 beta-glucosidase (1440 BGU/ml) were added tothe flask in an amount corresponding to 9 FPU/gram cellulose and 125BGU/gram cellulose. The small amount of glucose carried into the flaskwith the beta-glucosidase was taken into account.

Each flask was placed on a New Brunswick gyrotory shaker at 50° C. andshaken for 20 hours at 250 RPM. At the end of this period, the flaskcontents were filtered over glass micro fiber filter paper, and theglucose concentration in the filtrate was measured by a YSI glucoseanalyzer. The glucose concentration was related to the celluloseconcentration of the pretreated feedstock to determine the celluloseconversion.

FIG. 1 is a graph of cellulose conversion for certain feedstocks, as afunction of AX/NSP, at an average temperature of 121° C., according toEXAMPLE 3.

Surprisingly, as shown in FIG. 1, for this particular pretreatmentreaction the cellulose conversion increases linearly with the AX/NSP ofthe initial feedstock. The four feedstocks with the highest AX/NSP (oathulls and the three corn cobs) had the highest conversion to glucose.

These results indicate that the higher the AX/NSP of the feedstock, themore suitable the feedstock will be for ethanol production after a givenpretreatment.

EXAMPLE 4 High Performance Pretreatment Reaction with the Feedstocks

This example illustrates the comparative performance of the feedstocksafter a pretreatment reaction. This pretreatment reaction is atconditions that optimize performance in the subsequent cellulosehydrolysis.

Samples of 0.28 grams of Wiley-milled feedstocks from EXAMPLE 1 wereplaced in 7 grams of 1% sulfuric acid (pH 0.6 to 0.9) in a sealedstainless steel "bomb" reactor. The capacity of the bomb reactor is 9ml. For any one experiment, five bombs of identical contents were set upand the reaction products were combined to produce a pool of adequatequantity with which to work. The bombs were placed in a preheated 290°C. oil bath for 50 seconds, then removed and cooled by placing them intap water. Thermocouple measurements showed that the temperature in theinterior of the bomb reached 260° C. by the end of the heating period.The average equivalent temperature was 235° C.

The contents of the bombs were removed by rinsing with tap water, andthen vacuum-filtered over glass microfiber filter paper. The filtercakes were washed with tap water and air dried. The celluloseconcentration in the solids was determined by dissolution of aliquots in70% sulfuric acid, as described in EXAMPLE 1.

The reacted feedstocks were subjected to hydrolysis by cellulase asfollows. A sample of the reacted solids corresponding to 0.05 grams ofcellulose was added to a 25 ml flask with 4.9 grams of 0.05 M sodiumcitrate buffer, pH 4.8. Iogen Cellulase (140 FPU/ml) and Novozym 188beta-glucosidase (1440 BGU/ml) were added to the flask in an amountcorresponding to 9 FPU/gram cellulose and 125 BGU/gram cellulose. Thesmall amount of glucose carried into the flask with the beta-glucosidasewas taken into account.

Each flask was placed on an Orbit gyrotory shaker at 50° C. and shakenfor 20 hours at 250 RPM. At the end of this period, the contents of theflasks were filtered over glass microfiber filter paper, and the glucoseconcentration in the filtrate was measured by a DionexPulsed-Amperometric HPLC. The glucose concentration was related to thecellulose concentration in the pretreated feedstock to determine thecellulose conversion.

FIG. 2 is a graph of cellulose conversion for certain feedstocks, as afunction of AX/NSP, at an average temperature of 235° C., according toEXAMPLE 4.

As with the 121° C. reaction, FIG. 2 shows a cellulose conversion thatalso increases linearly with the AX/NSP of the initial feedstock. Thefour feedstocks with the highest AX/NSP (oat hulls and the three corncobs) had the highest level of cellulose conversion observed, with morethan 65% of the cellulose hydrolyzed to glucose.

These results demonstrate that the higher the AX/NSP of the feedstock,the more suitable the feedstock will be for ethanol production after ahigh performance pretreatment.

TABLE 5 shows the amount of cellulase enzyme required to reach 80%conversion to glucose. The amount of enzyme required is a key factor indetermining the feasibility of an ethanol production process. The datain TABLE 5 are derived from the results shown in FIG. 2 plus other datadescribing cellulose conversion as a function of cellulase dosage.

The top four feedstocks, including oat hulls and corn cobs, require 23%to 68% less cellulase enzyme to convert to cellulose to glucose than thenext best feedstock, wheat chaff. The top four feedstocks have a greatperformance advantage over the other feedstocks tested.

The top four feedstocks have AX/NSP greater than 0.39, while the otherfeedstocks have AX/NSP below this value. This data demonstrates thatsignificantly less cellulase enzyme is required for feedstocks withAX/NSP above about 0.39. This lower enzyme requirement is a significantadvantage in an ethanol production process.

                  TABLE 5    ______________________________________    CELLULASE ENZYME REQUIREMENTS                Cellulase dosage (FPU/g)    Feedstock   for 80% conversion in 20 hr                                AX/NSP    ______________________________________    Corn Cobs   6.6             0.420    (Red)    Corn cobs   8.7             0.412    (White)    Corn cobs   15.6            0.391    (Indian)    Oat hulls   16.3            0.422    Wheat chaff 21.0            0.376    Switch grass                27.1            0.357    Barley straw                28.3            0.317    Wheat straw 44.5            0.295    Maple wood  45.5            0.237    Corn stover 63.4            0.285    ______________________________________

EXAMPLE 5 Two-stage Pretreatment Reaction of Oat Hulls

This example demonstrates the use of a two-stage pretreatment reactionof oat hulls, the first mild stage followed by a second harsher stage.

For the first stage, samples of 4 grams of Wiley-milled feedstocks fromEXAMPLE 1 were placed in 96 grams of 1% sulfuric acid (pH 0.6 to 0.9) ina 250 ml flask. The contents of the flasks were gently mixed, and thenthe flasks were placed in a steam autoclave at 121° C. for 40 minutes.The flasks were then cooled and vacuum-filtered over glass microfiberfilter paper. The glucose, xylose, and arabinose concentrations of thefiltrates were determined by neutralizing with barium carbonate andanalyzing the samples by using a Dionex Pulsed-Amperometric HPLC. Thefilter cakes were washed with tap water and air dried. The cellulose,xylan, and arabinan concentrations in the solids were determined bydissolution of aliquots in 70% sulfuric acid, as described in EXAMPLE 1.The effect of the mild reaction on the cellulose and hemicellulose(arabinan+xylan) levels in the feedstock is shown in TABLE 6. Almost allof the hemicellulose is dissolved, which enriches the concentration ofcellulose.

                  TABLE 6    ______________________________________    COMPOSITION OF OAT HULLS AFTER MILD    PRETREATMENT REACTION    Feedstock: Oat hulls                  Cellulose (%)                             Hemicellulose (%)    ______________________________________    Before Pretreatment                  27.9       22.0    After Pretreatment                  39.5       3.0    ______________________________________

Samples of 0.28 grams of feedstocks reacted under mild conditions wereplaced in 7 grams of 1% sulfuric acid (pH 0.6 to 0.9) in a sealedstainless steel "bomb" reactor as described in EXAMPLE 4. Five bombs ofidentical contents were set up and the reaction products werecombined toproduce a pool of adequate quantity with which to work. The bombs wereplaced in a preheated 290° C. oil bath for 50 seconds, then removed andcooled by placing them in tap water.

The contents of the bombs were removed by rinsing with tap water, andthen vacuum-filtered over glass microfiber filter paper. The filtercakes were washed with tap water and air dried. The celluloseconcentration in the solids was determined by dissolution of aliquots in70% sulfuric acid, as described in EXAMPLE 1.

After one or two stages of pretreatment reaction, various feedstockswere subjected to hydrolysis by cellulase, as follows. A sample of thepretreated solids corresponding to 0.05 grams of cellulose was added toa 25 ml flask with 4.9 grams of 0.05 M sodium citrate buffer, pH 4.8.Iogen Cellulase (140 FPU/ml) and Novozym 188 beta-glucosidase (1440BGU/ml) were added to the flask in an amount corresponding to 10FPU/gram cellulose and 125 BGU per gram cellulose. The small amount ofglucose carried into the flask with the beta-glucosidase was taken intoaccount.

Each flask was placed on an Orbit gyrotory shaker at 50° C. and shakenfor 20 hours at 250 RPM. At the end of this period, the contents of theflasks were filtered over glass microfiber filter paper, and the glucoseconcentration in the filtrate was measured by a DionexPulsed-Amperometric HPLC. The glucose concentration was related to thecellulose concentration in the pretreated feedstock to determine theglucose yield.

The results are summarized in TABLE 7.

After the first stage of reaction, little hemicellulose remained in theoat hulls. The glucose yield after the cellulose was hydrolyzed bycellulase was only 340 mg/g.

After the second stage of pretreatment reaction, the glucose yield isover 85% higher than that of the first stage. The second stagepretreatment reaction therefore provided a significant enhancement ofthe hydrolysis performance. The two stage pretreatment results in aglucose yield within 6% of that after the single stage reaction of oathulls described in EXAMPLE 4.

These results ran exactly opposite to the teachings of Knappert, et al,who concluded that a material with low hemicellulose content does nothave an improved digestibility by cellulase enzymes after pretreatmentreaction. In the present example, after the first stage of reaction,very little hemicellulose remained in the oat hulls, yet the secondstage reaction increased the digestibility significantly. Knappert et altaught that such a low-hemicellulose material should not respond well topretreatment reaction. The present invention teaches the opposite.

                  TABLE 7    ______________________________________    TWO STAGE PRETREATMENT REACTION OF OAT HULLS                Hemicellulose    Pretreatment                content before this                             Glucose yield    reaction    stage (%)    (mg/g cellulose)    ______________________________________    Two stage   3.0          645    First stage 22.0         340    Single stage                22.0         685    (EXAMPLE 4)    ______________________________________

EXAMPLE 6 Large Scale Pretreatment Reaction with Oat Hulls

A large scale pretreatment of oat hulls was carried out using aWerner-Pflederer twin-screw extruder (Ramsey, N.J.). After milling in aWiley mill, the oat hulls were slurried to a 30% solids concentration in1% sulfuric acid (pH 0.7 to 1.2). The slurry was fed to the extruder ata rate of 10 pounds per hour and the pressure was 500 psig. The extruderwas maintained at 230° C. with live steam injection. At the average feedrate, the material passed through the extruder within 30 seconds. Theextruded oat hulls were collected and washed with water to removedissolved material, then filtered over glass microfiber filter paper.

The cellulose content of the extruded oat hulls was measured using themethods of EXAMPLE 1.

The extruded oat hulls were subjected to hydrolysis by cellulase asfollows. A sample of the extruded oat hulls corresponding to 0.05 gramsof cellulose was added to a 25 ml flask with 4.9 grams of 0.05 M sodiumcitrate buffer, pH 4.8. Iogen Cellulase (140 FPU/ml) and Novozym 188beta-glucosidase (1440 BGU/ml) were added to the flask in an amountcorresponding to 9 FPU/gram cellulose and 125 BGU/gram cellulose. Thesmall amount of glucose carried into the flask with the beta-glucosidasewas taken into account.

Each flask was placed on an Orbit gyrotory shaker at 50° C. and shakenfor 20 hours at 250 RPM. At the end of this period, the contents of theflask were filtered over glass microfiber filter paper, and the glucoseconcentration in the filtrate was measured by a DionexPulse-Amperometric HPLC. The glucose concentration was related to thecellulose concentration of the extruded oat hulls to determine theglucose yield.

The results are listed in TABLE 8. The glucose yield from the largescale pretreatment reaction of oat hulls was slightly (8%) less thanthat from the laboratory scale pretreatment in EXAMPLE 4. This indicatesthat the oat hull pretreatment reaction can be run on a large scale, asoptimization of the extrusion operation will no doubt overcome the 8%advantage of the laboratory pretreatment reaction.

                  TABLE 8    ______________________________________    GLUCOSE YIELD FROM PRETREATED OAT HULLS    Pretreatment   Glucose (mg/g cellulose)    ______________________________________    Extruder       630    Bomb (EXAMPLE 4)                   685    ______________________________________

EXAMPLE 7 Large Scale Pretreatment of Hardwood

A sample of aspen wood was pretreated using the steam explosion deviceand technique described by FOODY, U.S. Pat. No. 4,461,648. The resultingpretreated material was washed with water and is denoted as "Steamexploded hardwood". The cellulose content of the steam exploded hardwoodwas measured using the methods of EXAMPLE 1.

The steam exploded hardwood was subjected to hydrolysis by cellulaseenzyme as follows. A sample of the steam exploded hardwood correspondingto 0.05 grams of cellulose was added to a 25 ml flask with 4.9 grams of0.05 molar sodium citrate buffer, pH 4.8. Iogen Cellulase (140 FPU/ml)and Novozym 188 beta-glucosidase (1440 BGU/ml) were added to the flaskin an amount corresponding to 9 FPU/gram cellulose and 125 BGU/gramcellulose. The small amount of glucose carried into the flask with thebeta-glucosidase was taken into account.

Each flask was placed on an Orbit gyrotory shaker at 50° C. and shakenfor 20 hours at 250 RPM. At the end of this period, the contents of theflask were filtered over glass microfiber filter paper, and the glucoseconcentration in the filtrate was measured by a DionexPulsed-Amperometric HPLC. The glucose concentration was related to thecellulose concentration of the steam exploded hardwood to determine theglucose yield.

The results are listed in TABLE 9. The performance of the hardwoodreacted using the large scale device is within 2% by weight, of thatusing the laboratory device. In this case, the large scale use of steamexplosion has been extensively optimized and can match the laboratoryresults.

                  TABLE 9    ______________________________________    PRETREATMENT REACTION OF HARDWOOD    Device          Glucose yield (mg/g cellulose)    ______________________________________    Steam explosion 415    Laboratory (EXAMPLE 4)                    425    ______________________________________

EXAMPLE 8 Effect of Temperature on Single-stage and Two-stagePretreatment Reaction of Oat Hulls

This example demonstrates the use of a range of temperatures with bothsingle stage and two-stage pretreatment reactions of oat hulls.

For the single stage reactions, samples of 0.28 grams of oat hulls wereplaced in 7 grams of 1% sulfuric acid (pH 0.6) in a sealed stainlesssteel "bomb" reactor as described in EXAMPLE 4. Five bombs of identicalcontents were set up and the reaction products combined to produce apool of adequate quantity with which to work. The bombs were placed in apreheated oil bath, then removed and cooled by placing them in tapwater.

The temperatures and times in the oil bath were, as follows:

(1) 235° C., 50 seconds; (2) 180° C., 6 minutes; (3) 170° C., 8 minutes.

The contents of the bombs were removed by rinsing with tap water, andthen vacuum-filtered over glass microfiber filter paper. The filtercakes were washed with tap water and air dried. The celluloseconcentration in the solids was determined by dissolution of aliquots in70% sulfuric acid, as described in EXAMPLE 1.

For the two stage reactions, the first stage was carried out by placingsamples of 4 grams of Wiley-milled oat hulls in 96 grams of 1% sulfuricacid (pH 0.6) in a 250 ml flask. The contents of the flasks were gentlymixed, and then the flasks were placed in a steam autoclave at 121° C.for 40 minutes. The flasks were then cooled and the contents werevacuum-filtered over glass microfiber filter paper. The filter cakeswere washed with tap water and air dried. The cellulose, xylan, andarabinan concentrations in the solids were determined by dissolution ofaliquots in 70% sulfuric acid, as described in EXAMPLE 1.

The second stage was carried out by placing samples of 0.28 grams ofmaterial from the first stage in 7 grams of 1% sulfuric acid (pH 0.6) ina sealed stainless steel "bomb" reactor as described in EXAMPLE 4. Fivebombs of identical contents were set up and the reaction productscombined to produce a pool of adequate quantity to work with. The bombswere placed in a preheated oil bath, then removed and cooled by placingthem in tap water.

The temperatures and times in the oil bath matched those for the singlestage reaction: (1) 235° C., 50 seconds; (2) 180° C., 6 minutes; (3)170°C., 8 minutes.

The contents of the bombs were removed by rinsing with tap water, andthen vacuum-filtered over glass microfiber filter paper. The filtercakes were washed with tap water and air dried. The celluloseconcentration in the solids was determined by dissolution of aliquots in70% sulfuric acid, as described in EXAMPLE 1.

Feedstocks after one or two stages of reaction were subjected tocellulase enzyme hydrolysis as follows. A sample of the reacted solidscorresponding to 0.05 grams of cellulose was added to a 25 ml flask with4.9 grams of 0.05 molar sodium citrate buffer, pH 4.8. IOGEN Cellulase(140 FPU/ml) and NOVOZYM 188 beta-glucosidase (1440 BGU/ml) were addedto the flask in an amount corresponding to 9 FPU/gram cellulose and 125BGU per gram cellulose. The small amount of glucose carried into theflask with the beta-glucosidase was taken into account.

Each flask was placed on an Orbit gyrotory shaker at 50° C. and shakenfor 20 hours at 250 RPM. At the end of this period, the contents of theflasks were filtered over glass microfiber filter paper, and the glucoseconcentration in the filtrate was measured by a DionexPulsed-Amperometric HPLC. The glucose concentration was related to thecellulose concentration in the pretreated feedstock to determine theglucose yield.

The results are summarized in TABLE 10.

Using a single stage reaction, the glucose yield is almost as high at180° C. as at the optimum temperature. The glucose yield drops as thetemperature is decreased from 180° C. to 170 C.

The two stage reaction has the same temperature profile as the singlestage pretreatment reaction, with a similar performance at 180° C. andthe optimum temperature, and a drop in performance below 180° C. Glucoseyields in the two-stage reaction were 15% below those with the singlestage reaction.

                  TABLE 10    ______________________________________    EFFECT OF TEMPERATURE ON GLUCOSE YIELD FROM    OAT HULLS                                           Relative            Reaction    Reaction Glucose yield                                           Glucose    Pretreatment            Temperature (C.)                        Time (sec)                                 (mg/g cellulose)                                           yield    ______________________________________    Single stage            235         50       685       100    Single stage            180         360      660       96    Single stage            170         480      555       81    Two stages             235*       50       575       84    Two stages             180*       360      560       82    Two stages             170*       480      485       71    ______________________________________     *Following a first stage at 121 C.

While preferred embodiments of our invention have been shown anddescribed, the invention is to be defined solely by the scope of theappended claims, including any equivalent for each recited claim elementthat would occur to one of ordinary skill and would not be precluded byprior art considerations.

We claim:
 1. An improved process for pretreating a lignocellulosicfeedstock intended to be converted to fuel ethanol, consistingessentially of the following steps:a. Choosing a feedstock comprised ofat least hemicellulose and cellulose, and characterized by a ratio ofarabinan plus xylan to total nonstarch polysaccharides (AX/NSP) that isgreater than 0.39, wherein the feedstock consists of oat hulls or corncobs; and b. Reacting the chosen feedstock at conditions which disruptits fiber structure and effect an hydrolysis of a portion of thehemicellulose and cellulose, in order to create a pretreated feedstockwith increased accessibility to being digested, during a treatment withcellulase enzymes.
 2. A process according to claim 1, wherein thereaction step is carried out by subjecting the chosen feedstock at anaverage temperature of 180° C. to 270° C., pH 0.5 to 2.5 for a period of5 seconds to 60 minutes.
 3. A process according to claim 2, wherein thereaction step is carried out by subjecting the chosen feedstock at anaverage temperature of 220° C. to 270° C., pH 0.5 to 2.5 for a period of5 seconds to 120 seconds.
 4. A process according to claim 1, wherein thereaction step is carried out in two stages, with the first stage at anaverage temperature below 180° C. and the second stage at an averagetemperature above 180° C.
 5. A process according to claim 4, wherein thefirst stage of the reaction step is carried out at an averagetemperature of 60° C. to 140° C. for 0.25 to 24 hours at pH 0.5 to 2.5.6. A process according to claim 5, wherein the first stage of thereaction step is carried out at an average temperature of 100° C. to130° C. for 0.5 to 3 hours at pH 0.5 to 2.5.
 7. A process according toclaim 4, wherein the second stage of the reaction step is carried out bysubjecting the chosen feedstock to an average temperature of 180° C. to270° C., at pH 0.5 to 2.5, for a period of 5 seconds to 60 minutes.
 8. Aprocess according to claim 7, wherein the second stage of the reactionstep is carried out by subjecting the chosen feedstock at an averagetemperature of 220° C. to 270° C., at pH 0.5 to 2.5 for a period of 5seconds to 120 seconds.
 9. A process according to claim 1, wherein thefeedstock is slurried in water during the reaction step.
 10. A processaccording to claim 1, wherein the feedstock is free of externally-addedmoisture during the reaction step.
 11. A process according to claim 1,wherein the reaction step is carried out with a steam explosion orextrusion device.
 12. An improved process for converting alignocellulosic feedstock to ethanol, consisting essentially of thefollowing steps:a. Choosing a feedstock comprised of at leasthemicellulose and cellulose, and characterized by a ratio of arabinanplus xylan to total nonstarch polysaccharides (AX/NSP) that is greaterthan about 0.39, wherein the feedstock consists of oat hulls or corncobs; and b. Reacting the chosen feedstock at conditions which disruptits fiber structure and effect an hydrolysis of a portion of thehemicellulose and cellulose, in order to create a pretreated feedstockwith increased accessibility to being digested, during a treatment withcellulase enzymes; and c. Hydrolyzing the pretreated feedstock by usingcellulase enzymes; and d. Fermenting the resulting sugars to ethanol;and e. Recovering the ethanol.
 13. A process according to claim 12wherein the reaction step is carried out by subjecting the chosenfeedstock to an average temperature of 180° C. to 270° C., at pH 0.5 to2.5, for a period of 5 seconds to 60 minutes, and the subsequentenzymatic hydrolysis of cellulose is carried out with 1.0 to 10.0 filterpaper units (FPU) of cellulase enzyme per gram of cellulose.
 14. Aprocess according to claim 13 wherein the reaction step is carried outby subjecting the chosen feedstock to an average temperature of 220° C.to 270° C., at pH 0.5 to 2.5 for a period of 5 seconds to 120 seconds,and the subsequent enzymatic hydrolysis of cellulose is carried out with1.0 to 10.0 filter paper units (FPU) of cellulase enzyme per gram ofcellulose.
 15. A process according to claim 12 wherein the reaction stepis carried out in two stages, with the first stage at an averagetemperature of below 180° C. and the second stage at an averagetemperature of above 180° C.
 16. A process according to claim 15 whereinthe first stage of the reaction step is carried out at an averagetemperature of 60° C. to 140° C. for 0.25 to 24 hours at pH 0.5 to 2.5.17. A process according to claim 16 wherein the first stage of thereaction step is carried out at an average temperature of 100° C. to130° C. for 0.5 to 3 hours at pH 0.5 to 2.5.
 18. A process according toclaim 15 wherein the second stage of the reaction step is carried out bysubjecting the chosen feedstock at an average temperature of 180° C. to270° C., at pH 0.5 to 2.5, for a period of 5 seconds to 60 minutes, andthe subsequent enzymatic hydrolysis of cellulose is carried out with 1.0to 10.0 filter paper units (FPU) of cellulase enzyme per gram ofcellulose.
 19. A process according to claim 15 wherein the second stageof the reaction step is carried out by subjecting the chosen feedstockat an average temperature of 220° C. to 270° C., at pH 0.5 to 2.5, for aperiod of 5 seconds to 120 seconds, and the subsequent enzymatichydrolysis of cellulose is carried out with 1.0 to 10.0 filter paperunits (FPU) of cellulase enzyme per gram of cellulose.
 20. A processaccording to claim 12, wherein the feedstock is slurried in water duringthe reaction step.
 21. A process according to claim 12, wherein thefeedstock is free of externally-added moisture during the reaction step.22. A process according to claim 12, wherein the pretreatment step iscarried out with a steam explosion or extrusion device.
 23. A processaccording to claim 12, wherein the cellulose hydrolysis step and ethanolfermentation step are carried out simultaneously.
 24. An improvedprocess for converting a lignocellulosic feedstock to glucose,consisting essentially of the following steps:a. Choosing a feedstockcomprised of at least hemicellulose and cellulose, and characterized bya ratio of arabinan plus xylan to total nonstarch polysaccharides(AX/NSP) of greater than about 0.39, wherein the feedstock consists ofcorn cobs or oat hulls; and b. Reacting the chosen feedstock atconditions which disrupt its fiber structure and effect an hydrolysis ofa portion of the hemicellulose and cellulose, in order to create apretreated feedstock with increased accessibility to being digested,during a treatment with cellulase enzymes; and c. Hydrolyzing at least40% of the cellulose in the pretreated feedstock to glucose by usingcellulase enzymes.
 25. A process according to claim 24 wherein thereaction step is carried out by subjecting the chosen feedstock to anaverage temperature of 180° C. to 270° C., at pH 0.5 to 2.5, for aperiod of 5 seconds to 60 minutes, and the subsequent enzymatichydrolysis of cellulose is carried out with 1.0 to 10.0 filter paperunits (FPU) of cellulase enzyme per gram of cellulose.
 26. A processaccording to claim 24 wherein the reaction step is carried out bysubjecting the chosen feedstock to an average temperature of 220° C. to270° C., at pH 0.5 to 2.5, for a period of 5 seconds to 120 seconds, andthe subsequent enzymatic hydrolysis of cellulose is carried out with 1.0to 10.0 filter paper units (FPU) of cellulase enzyme per gram ofcellulose.
 27. A process according to claim 24 wherein the reaction stepis carried out in two stages, with the first stage at an averagetemperature below 180° C. and the second stage at an average temperatureabove 180° C.
 28. A process according to claim 27 wherein the firststage of the reaction step is carried out at an average temperature ot60° C. to 140° C. for 0.25 to 24 hours at pH 0.5 to 2.5.
 29. A processaccording to claim 27 wherein the first stage of the reaction step iscarried out at an average temperature of 100° C. to 130° C. for 0.5 to 3hours at pH 0.5 to 2.5.
 30. A process according to claim 27 wherein thesecond stage of the reaction step is carried out by subjecting thechosen feedstock to an average temperature of 180° C. to 270° C., at pH0.5 to 2.5, for a period of 5 seconds to 60 minutes, and the subsequentenzymatic hydrolysis of cellulose is carried out with 1.0 to 10.0 filterpaper units (FPU) of cellulase enzyme per gram of cellulose.
 31. Aprocess according to claim 27 wherein the second stage of the reactionstep is carried out by subjecting the chosen feedstock to an averagetemperature of 220° C. to 270° C., at pH 0.5 to 2.5, for a period of 5seconds to 120 seconds, and the subsequent enzymatic hydrolysis ofcellulose is carried out with 1.0 to 10.0 filter paper units (FPU) ofcellulase enzyme per gram of cellulose.
 32. A process according to claim24, wherein the feedstock is slurried in water during the reaction step.33. A process according to claim 24, wherein the feedstock is free ofexternally-added moisture during the reaction step.
 34. A processaccording to claim 24, wherein the reaction step is carried out with asteam explosion or extrusion device.
 35. An improved process forpretreating a lignocellulosic feedstock for conversion to fuel ethanol,consisting essentially of the following steps:a. Choosing a selectivelybred feedstock, comprised of at least hemicellulose and cellulose, onthe basis of an increased ratio of arabinan plus xylan to totalnonstarch polysaccharides (AX/NSP) over a starting material to a levelgreater than about 0.39; and b. Reacting the selectively bred feedstockat an average temperature of 180° C. to 270° C., at pH 0.5 to 2.5, for aperiod of 5 seconds to 60 minutes to disrupt the fiber structure andeffect an hydrolysis of a portion of the hemicellulose and cellulose, inorder to create a pretreated feedstock with increased accessibility tobeing digested, during a treatment with cellulase enzymes.
 36. A processaccording to claim 35 wherein the reaction step is carried out bysubjecting the selectively bred feedstock to an average temperature of220° C. to 270° C., at pH 0.5 to 2.5 for a period of 5 seconds to 120seconds.
 37. An improved process for pretreating a lignocellulosicfeedstock for conversion to fuel ethanol, consisting essentially of thefollowing steps:a. Choosing a selectively bred feedstock, comprised ofat least hemicellulose and cellulose, on the basis of an increased ratioof arabinan plus xylan to total nonstarch polysaccharides (AX/NSP) overa starting feedstock material to a level that is greater than about0.39; and b. Reacting the selectively bred feedstock in two stages, withthe first stage at an average temperature below 180 C. and the secondstage at an average temperature above 180 C. to disrupt the fiberstructure and effect an hydrolysis of a portion of the hemicellulose andcellulose, in order to create a pretreated feedstock with increasedaccessibility to being digested, during a treatment with cellulaseenzymes.
 38. A process according to claim 37 wherein the first stage ofthe reaction step is carried out at an average temperature of 60° C. to140° C. for 0.25 to 24 hours at pH 0.5 to 2.5.
 39. A process accordingto claim 38 wherein the first stage-of the reaction step is carried outat an average temperature of 100° C. to 130° C. for 0.5 to 3 hours at pH0.5 to 2.5.
 40. A process according to claim 37 wherein the second stageof the reaction step is carried out by subjecting the selectively bredfeedstock to an average temperature of 180° C. to 270° C., at pH 0.5 to2.5, for a period of 5 seconds to 60 minutes.
 41. A process according toclaim 40 wherein the second stage of the reaction step is carried out bysubjecting the chosen feedstock at an average temperature of 220° C. to270° C., at pH 0.5 to 2.5, for a period of 5 seconds to 120 seconds. 42.A process according to claim 35 wherein the pretreatment is carried outwith a steam explosion or extrusion device during the reaction step. 43.A process for converting a lignocellulosic feedstock to ethanol, theprocess consisting essentially of the following steps:a. Choosing aselectively bred feedstock, comprised of at least hemicellulose andcellulose, on the basis of an increased ratio of arabinan plus xylan tototal nonstarch polysaccharides (AX/NSP) over a starting feedstockmaterial to a level that is greater than about 0.39; and b. Reacting theselectively bred feedstock at an average temperature of 180° C. to 270°C., at pH 0.5 to 2.5, for a period of 5 seconds to 60 minutes to disruptthe fiber structure and effect an hydrolysis of a portion of thehemicellulose and cellulose, in order to create a pretreated feedstockwith increased accessibility to being digested, during a treatment withcellulase enzymes; and c. Hydrolyzing the pretreated feedstock to sugarsby using cellulase enzymes at a dosage of 1.0 to 10.0 filter paper units(FPU) of cellulase enzyme per gram of cellulose; and d. Fermenting theresulting sugars to ethanol; and e. Recovering the ethanol.
 44. Aprocess according to claim 43 wherein the reaction step is carried outby subjecting the selectively bred feedstock to an average temperatureof 220° C. to 270° C., at pH 0.5 to 2.5, for a period of 5 seconds to120 seconds.
 45. A process for converting a lignocellulosic feedstock toethanol, the process consisting essentially of the following steps:a.Choosing a selectively bred feedstock, comprised of at leasthemicellulose and cellulose, on the basis of an increased ratio ofarabinan plus xylan to total nonstarch polysaccharides (AX/NSP) over astarting feedstock material to a level that is greater than about 0.39;and b. Reacting the chosen feedstock in two stages, with the first stageat an average temperature below 180° C. and the second stage at anaverage temperature above 180 C. to disrupt the fiber structure andeffect an hydrolysis of a portion of the hemicellulose and cellulose, inorder to create a pretreated feedstock with increased accessibility tobeing digested, during a treatment with cellulase enzymes; and c.Hydrolyzing the pretreated feedstock to sugars by using cellulaseenzymes at a dosage of 1.0 to 10.0 filter paper units (FPU) of cellulaseenzyme per gram of cellulose; and d. Fermenting the resulting sugars toethanol; and e. Recovering the ethanol.
 46. A process according to claim45 wherein the first stage of the reaction step is carried out at anaverage temperature of 60° C. to 140° C. for 0.25 to 24 hours at pH 0.5to 2.5.
 47. A process according to claim 46 wherein the first stage ofthe reaction step is carried out at an average temperature of 110° C. to130° C. for 0.5 to 3 hours at pH 0.5 to 2.5.
 48. A process according toclaim 45 wherein the second stage of the reaction step is carried out bysubjecting the bred feedstock to an average temperature of 180° C. to270° C., at pH 0.5 to 2.5, for a period of 5 seconds to 60 minutes. 49.A process according to claim 48 wherein the second stage of the reactionstep is carried out by subjecting the selectively bred feedstock to anaverage temperature of 220° C. to 270° C., at pH 0.5 to 2.5, for aperiod of 5 seconds to 120 seconds.
 50. A process according to claim 43wherein the reaction step is carried out with a steam explosion orextrusion device.
 51. A process for converting a lignocellulosicfeedstock to glucose, consisting essentially of the following steps:a.Choosing a selectively bred feedstock, comprised of at leasthemicellulose and cellulose, on the basis of an increased ratio ofarabinan plus xylan to total nonstarch polysaccharides (AX/NSP) over astarting feedstock material to a level that is greater than about 0.39;and b. Reacting the selectively bred feedstock at an average temperatureof 180° C. to 270° C., at a pH of 0.5 to 2.5, for a period of 5 secondsto 60 minutes to disrupt the fiber structure and effect an hydrolysis ofa portion of the hemicellulose and cellulose, in order to create apretreated feedstock with increased accessibility to being digested,during a treatment with cellulase enzymes; and c. Hydrolyzing at least40% of the cellulose in the pretreated feedstock to glucose by usingcellulase enzymes at a dosage of 1.0 to 10.0 filter paper units (FPU) ofcellulase enzyme per gram of cellulose.
 52. A process according to claim51 wherein the reaction step is carried out by subjecting theselectively bred feedstock to an average temperature of 220° C. to 270°C., at a pH of 0.5 to 2.5, for a period of 5 seconds to 120 seconds. 53.A process for converting a lignocellulosic feedstock to glucose,consisting essentially of the following steps:a. Choosing a selectivelybred feedstock, comprised of at least hemicellulose and cellulose, onthe basis of an increased ratio of arabinan plus xylan to totalnonstarch polysaccharides (AX/NSP) over a starting feedstock material toa level greater than about 0.39; and b. Reacting the selectively bredfeedstock in two stages, with the first stage at an average temperaturebelow 180° C. and the second stage at an average temperature above 180C. to disrupt the fiber structure and effect an hydrolysis of a portionof the hemicellulose and cellulose, in order to create a pretreatedfeedstock with increased accessibility to being digested, during atreatment with cellulase enzymes; and c. Hydrolyzing at least 40% of thecellulose in the pretreated feedstock to glucose by using cellulaseenzymes at a dosage of 1.0 to 10.0 filter paper units (FPU) of cellulaseenzyme per gram of cellulose.
 54. A process according to claim 53wherein the first stage of the reaction step is carried out at anaverage temperature of 60° C. to 140° C. for 0.25 to 24 hours at pH 0.5to 2.5.
 55. A process according to claim 54 wherein the first stage ofthe reaction step is carried out at an average temperature of 110° C. to130° C. for 0.5 to 3 hours at pH 0.5 to 2.5.
 56. A process according toclaim 53 wherein the second stage of the reaction step is carried out bysubjecting the selectively bred feedstock to an average temperature of180° C. to 270° C., at pH 0.5 to 2.5, for a period of 5 seconds to 60minutes.
 57. A process according to claim 56 wherein the second stage-ofthe reaction step is carried out by subjecting the chosen feedstock toan average temperature of 220° C. to 270° C., at pH 0.5 to 2.5, for aperiod of 5 seconds to 120 seconds.
 58. The process according to claim51 wherein the reaction step is carried out with a steam explosion orextrusion device.